Proinflammatory high-density lipoprotein results from oxidized lipid mediators in the pathogenesis of both idiopathic and associated types of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by abnormal elaboration of vasoactive peptides, endothelial cell dysfunction, vascular remodeling, and inflammation, which collectively contribute to its pathogenesis. We investigated the potential for high-density lipoprotein (HDL) dysfunction (i.e., proinflammatory effects) and abnormal plasma eicosanoid levels to contribute to the pathobiology of PAH and assessed ex vivo the effect of treatment with apolipoprotein A-I mimetic peptide 4F on the observed HDL dysfunction. We determined the "inflammatory indices" HII and LII for HDL and low-density lipoprotein (LDL), respectively, in subjects with idiopathic PAH (IPAH) and associated PAH (APAH) by an in vitro monocyte chemotaxis assay. The 4F was added ex vivo, and repeat LII and HII values were obtained versus a sham treatment. We further determined eicosanoid levels in plasma and HDL fractions from patients with IPAH and APAH relative to controls. The LIIs were significantly higher for IPAH and APAH patients than for controls. Incubation of plasma with 4F before isolation of LDL and HDL significantly reduced the LII values, compared with sham-treated LDL, for IPAH and APAH. The increased LII values reflected increased states of LDL oxidation and thereby increased proinflammatory effects in both cohorts. The HIIs for both PAH cohorts reflected a "dysfunctional HDL phenotype," that is, proinflammatory HDL effects. In contrast to "normal HDL function," the determined HIIs were significantly increased for the IPAH and APAH cohorts. Ex vivo 4F treatment significantly improved the HDL function versus the sham treatment. Although there was a significant "salutary effect" of 4F treatment, this did not entirely normalize the HII. Significantly increased levels for both IPAH and APAH versus controls were evident for the eicosanoids 9-HODE, 13-HODE, 5-HETE, 12-HETE, and 15-HETE, while no statistical differences were evident for comparisons of IPAH and APAH for the determined plasma eicosanoid levels in the HDL fractions. Our study has further implicated the putative role of "oxidant stress" and inflammation in the pathobiology of PAH. Our data suggest the influences on the "dysfunctional HDL phenotype" of increased oxidized fatty acids, which are paradoxically proinflammatory. We speculate that therapies that target either the "inflammatory milieu" or the "dysfunctional HDL phenotype," such as apoA-I mimetic peptides, may be valuable avenues of further research in pulmonary vascular diseases.

Isolevuglandin-type lipid aldehydes induce the inflammatory response of macrophages by modifying phosphatidylethanolamines and activating the receptor for advanced glycation endproducts.

AIMS: Increased lipid peroxidation occurs in many conditions associated with inflammation. Because lipid peroxidation produces lipid aldehydes that can induce inflammatory responses through unknown mechanisms, elucidating these mechanisms may lead to development of better treatments for inflammatory diseases. We recently demonstrated that exposure of cultured cells to lipid aldehydes such as isolevuglandins (IsoLG) results in the modification of phosphatidylethanolamine (PE). We therefore sought to determine (i) whether PE modification by isolevuglandins (IsoLG-PE) occurred in vivo, (ii) whether IsoLG-PE stimulated the inflammatory responses of macrophages, and (iii) the identity of receptors mediating the inflammatory effects of IsoLG-PE. RESULTS: IsoLG-PE levels were elevated in plasma of patients with familial hypercholesterolemia and in the livers of mice fed a high-fat diet to induce obesity and hepatosteatosis. IsoLG-PE potently stimulated nuclear factor kappa B (NFκB) activation and expression of inflammatory cytokines in macrophages. The effects of IsoLG-PE were blocked by the soluble form of the receptor for advanced glycation endproducts (sRAGE) and by RAGE antagonists. Furthermore, macrophages derived from the bone marrow of Ager null mice failed to express inflammatory cytokines in response to IsoLG-PE to the same extent as macrophages from wild-type mice. INNOVATION: These studies are the first to identify IsoLG-PE as a mediator of macrophage activation and a specific receptor, RAGE, which mediates its biological effects. CONCLUSION: PE modification by IsoLG forms RAGE ligands that activate macrophages, so that the increased IsoLG-PE generated by high circulating cholesterol levels or high-fat diet may play a role in the inflammation associated with these conditions.

Transgenic 6F tomatoes act on the small intestine to prevent systemic inflammation and dyslipidemia caused by Western diet and intestinally derived lysophosphatidic acid.

We recently reported that levels of unsaturated lysophosphatidic acid (LPA) in the small intestine significantly correlated with the extent of aortic atherosclerosis in LDL receptor-null (LDLR⁻/⁻) mice fed a Western diet (WD). Here we demonstrate that WD increases unsaturated (but not saturated) LPA levels in the small intestine of LDLR⁻/⁻ mice and causes changes in small intestine gene expression. Confirmation of microarray analysis by quantitative RT-PCR showed that adding transgenic tomatoes expressing the apoA-I mimetic peptide 6F (Tg6F) to WD prevented many WD-mediated small intestine changes in gene expression. If instead of feeding WD, unsaturated LPA was added to chow and fed to the mice: i) levels of LPA in the small intestine were similar to those induced by feeding WD; ii) gene expression changes in the small intestine mimicked WD-mediated changes; and iii) changes in plasma serum amyloid A, total cholesterol, triglycerides, HDL-cholesterol levels, and the fast-performance liquid chromatography lipoprotein profile mimicked WD-mediated changes. Adding Tg6F (but not control tomatoes) to LPA-supplemented chow prevented the LPA-induced changes. We conclude that: i) WD-mediated systemic inflammation and dyslipidemia may be in part due to WD-induced increases in small intestine LPA levels; and ii) Tg6F reduces WD-mediated systemic inflammation and dyslipidemia by preventing WD-induced increases in LPA levels in the small intestine.

A novel approach to oral apoA-I mimetic therapy.

Transgenic tomato plants were constructed with an empty vector (EV) or a vector expressing an apoA-I mimetic peptide, 6F. EV or 6F tomatoes were harvested, lyophilized, ground into powder, added to Western diet (WD) at 2.2% by weight, and fed to LDL receptor-null (LDLR(-/-)) mice at 45 mg/kg/day 6F. After 13 weeks, the percent of the aorta with lesions was 4.1 ± 4%, 3.3 ± 2.4%, and 1.9 ± 1.4% for WD, WD + EV, and WD + 6F, respectively (WD + 6F vs. WD, P = 0.0134; WD + 6F vs. WD + EV, P = 0.0386; WD + EV vs. WD, not significant). While body weight did not differ, plasma serum amyloid A (SAA), total cholesterol, triglycerides, and lysophosphatidic acid (LPA) levels were less in WD + 6F mice; P < 0.0295. HDL cholesterol and paroxonase-1 activity (PON) were higher in WD + 6F mice (P = 0.0055 and P = 0.0254, respectively), but not in WD + EV mice. Plasma SAA, total cholesterol, triglycerides, LPA, and 15-hydroxyeicosatetraenoic acid (HETE) levels positively correlated with lesions (P < 0.0001); HDL cholesterol and PON were inversely correlated (P < 0.0001). After feeding WD + 6F: i) intact 6F was detected in small intestine (but not in plasma); ii) small intestine LPA was decreased compared with WD + EV (P < 0.0469); and iii) small intestine LPA 18:2 positively correlated with the percent of the aorta with lesions (P < 0.0179). These data suggest that 6F acts in the small intestine and provides a novel approach to oral apoA-I mimetic therapy.

High-density lipoprotein and 4F peptide reduce systemic inflammation by modulating intestinal oxidized lipid metabolism: novel hypotheses and review of literature.

Oxidized phospholipids are found in the vasculature of animal models of atherosclerosis, in human atherosclerotic lesions, and in other inflammatory diseases. Oxidized phospholipids cause vascular and nonvascular cells to initiate an inflammatory reaction. Metabolites of arachidonic acid, such as 12-hydroxyeicosatetraenoic acid, can mimic some of the inflammatory properties of oxidized phospholipids. In vitro and in vivo normal high-density lipoprotein (HDL), normal apolipoprotein A-I, and apolipoprotein A-I mimetic peptides, each likely acting in a different manner, prevent the inflammatory reaction characteristic of atherosclerosis, and this is associated with decreased levels of oxidized lipids in tissues and cells. HDL from animal models of atherosclerosis or from humans with atherosclerosis or from humans or animals with other chronic inflammatory diseases does not prevent the inflammatory reaction characteristic of atherosclerosis and may even enhance the inflammatory reaction. In mice and perhaps humans, ≈30% of the steady-state plasma HDL-cholesterol pool is derived from the small intestine. The metabolism of phospholipids by gut bacteria has been recently implicated in atherosclerosis in both mice and humans. Studies with apolipoprotein A-I mimetic peptides suggest that the small intestine is a major tissue regulating systemic inflammation in mouse models of atherosclerosis and may be important for determining the functionality of HDL.

LXRα is uniquely required for maximal reverse cholesterol transport and atheroprotection in ApoE-deficient mice.

The liver X receptor (LXR) signaling pathway is an important modulator of atherosclerosis, but the relative importance of the two LXRs in atheroprotection is incompletely understood. We show here that LXRα, the dominant LXR isotype expressed in liver, plays a particularly important role in whole-body sterol homeostasis. In the context of the ApoE(-/-) background, deletion of LXRα, but not LXRß, led to prominent increases in atherosclerosis and peripheral cholesterol accumulation. However, combined loss of LXRα and LXRß on the ApoE(-/-) background led to an even more severe cholesterol accumulation phenotype compared to LXRα(-/-)ApoE(-/-) mice, indicating that LXRß does contribute to reverse cholesterol transport (RCT) but that this contribution is quantitatively less important than that of LXRα. Unexpectedly, macrophages did not appear to underlie the differential phenotype of LXRα(-/-)ApoE(-/-) and LXRß(-/-)ApoE(-/-) mice, as in vitro assays revealed no difference in the efficiency of cholesterol efflux from isolated macrophages. By contrast, in vivo assays of RCT using exogenously labeled macrophages revealed a marked defect in fecal sterol efflux in LXRα(-/-)ApoE(-/-) mice. Mechanistically, this defect was linked to a specific requirement for LXRα(-/-) in the expression of hepatic LXR target genes involved in sterol transport and metabolism. These studies reveal a previously unrecognized requirement for hepatic LXRα for optimal reverse cholesterol transport in mice.

HDL and cardiovascular disease: atherogenic and atheroprotective mechanisms.

The lipoprotein HDL has two important roles: first, it promotes reverse cholesterol transport, and second, it modulates inflammation. Epidemiological studies show that HDL-cholesterol levels are inversely correlated with the risk of cardiovascular events. However, many patients who experience a clinical event have normal, or even high, levels of HDL cholesterol. Measuring HDL-cholesterol levels provides information about the size of the HDL pool, but does not predict HDL composition or function. The main component of HDL, apolipoprotein A-I (apo A-I), is largely responsible for reverse cholesterol transport through the macrophage ATP-binding cassette transporter ABCA1. Apo A-I can be damaged by oxidative mechanisms, which render the protein less able to promote cholesterol efflux. HDL also contains a number of other proteins that are affected by the oxidative environment of the acute-phase response. Modification of the protein components of HDL can convert it from an anti-inflammatory to a proinflammatory particle. Small peptides that mimic some of the properties of apo A-I have been shown in preclinical models to improve HDL function and reduce atherosclerosis without altering HDL-cholesterol levels. Robust assays to evaluate the function of HDL are needed to supplement the measurement of HDL-cholesterol levels in the clinic.

Apolipoprotein A-I (apoA-I) and apoA-I mimetic peptides inhibit tumor development in a mouse model of ovarian cancer.

We examined whether reduced levels of Apolipoprotein A-I (apoA-I) in ovarian cancer patients are causal in ovarian cancer in a mouse model. Mice expressing a human apoA-I transgene had (i) increased survival (P < 0.0001) and (ii) decreased tumor development (P < 0.01), when compared with littermates, following injection of mouse ovarian epithelial papillary serous adenocarcinoma cells (ID-8 cells). ApoA-I mimetic peptides reduced viability and proliferation of ID8 cells and cis-platinum-resistant human ovarian cancer cells, and decreased ID-8 cell-mediated tumor burden in C57BL/6J mice when administered subcutaneously or orally. Serum levels of lysophosphatidic acid, a well-characterized modulator of tumor cell proliferation, were significantly reduced (>50% compared with control mice, P < 0.05) in mice that received apoA-I mimetic peptides (administered either subcutaneously or orally), suggesting that binding and removal of lysophosphatidic acid is a potential mechanism for the inhibition of tumor development by apoA-I mimetic peptides, which may serve as a previously unexplored class of anticancer agents.

L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL in mice.

To determine in vivo if L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL. Injecting L-4F into apoE null mice resulted in a significant reduction in plasma levels of 15-HETE, 5-HETE, 13-HODE and 9-HODE. In contrast, plasma levels of 20-HETE were not reduced and plasma levels of 14,15-EET, which are derived from the cytochrome P450 pathway, were elevated after injection of L-4F. Injection of 13(S)-HPODE into wild-type C57BL/6J mice caused an increase in plasma levels of 13-HODE and 9-HODE and was accompanied by a significant loss in the anti-inflammatory properties of HDL. The response of atherosclerosis resistant C3H/HeJ mice to injection of 13(S)-HPODE was similar but much more blunted. Injection of L-4F at a site different from that at which the 13(S)-HPODE was injected resulted in significantly lower plasma levels of 13-HODE and 9-HODE and significantly less loss of HDL anti-inflammatory properties in both strains. i) L-4F differentially alters plasma levels of oxidized fatty acids in vivo. ii) The resistance of the C3H/HeJ strain to atherosclerosis may in part be mediated by a reduced reaction of this strain to these potent lipid oxidants.

Structure and function of HDL mimetics.

HDL mimetics have been constructed from a number of peptides and proteins with varying structures, all of which bind lipids found in HDL. HDL mimetics containing a peptide or protein have been constructed with as few as 4 and as many as 243 amino acid residues. Some HDL mimetics have been constructed with lipid but without a peptide or protein component. Some HDL mimetics promote cholesterol efflux, some have been shown to have a remarkable ability to bind oxidized lipids compared to human apolipoprotein A-I (apoA-I). Many of these peptides have been shown to have antiinflammatory properties. Based on studies in a number of animal models and in early human clinical trials, HDL mimetics appear to have promise as diagnostic and therapeutic agents.

The role of dysfunctional HDL in atherosclerosis.

This review focuses on HDL function in modulating LDL oxidation and LDL-induced inflammation. Dysfunctional HDL has been identified in animal models and humans with chronic inflammatory diseases including atherosclerosis. The loss of antiinflammatory function correlated with a loss of function in reverse cholesterol transport. In animal models and perhaps in humans, dysfunctional HDL can be improved by apoA-I mimetic peptides that bind oxidized lipids with high affinity.

Apolipoprotein A-I mimetic peptides.

Recent publications reveal the mechanism of action of apolipoprotein A-I (apoA-I) mimetic peptides to be the remarkable binding affinity that oxidized lipids have for these peptides compared with apoA-I. There was no difference in the binding affinity of oxidized lipids or in peptide efficacy in reducing inflammation and atherosclerosis in rabbits injected with peptides synthesized from all D- or all L-amino acids. The apoA-I mimetic peptide 4F increased the formation of pre-beta high-density lipoprotein, increased cholesterol efflux, and reduced lipoprotein oxidation in vitro; it increased antioxidants and vascular repair in type 1 diabetic rats; it improved vasodilation, oxidative stress, myocardial inflammation, and angiogenic potential in a mouse model of scleroderma; it reduced renal inflammation in low-density lipoprotein receptor-null mice fed a Western diet; it reduced arthritis in a rat model; it reduced adiposity, increased adiponectin levels, and improved insulin sensitivity in obese mice; and it improved high-density lipoprotein inflammatory properties in humans with coronary heart disease.

Multiple indications for anti-inflammatory apolipoprotein mimetic peptides.

Apolipoprotein mimetic peptides dramatically reduce atherosclerosis in animal models, and may be an excellent mode of therapy to treat a variety of vascular inflammatory conditions, including atherosclerosis. Studies of apolipoprotein mimetic peptides in models of inflammatory disorders other than atherosclerosis, including viral influenza, asthma, chronic rejection after heart transplantation, sickle cell disease, scleroderma, diabetes, cognitive dysfunction and renal inflammation, suggest that apolipoprotein mimetic peptides may have efficacy in a wide range of inflammatory conditions.

Anti-inflammatory apoA-I-mimetic peptides bind oxidized lipids with much higher affinity than human apoA-I.

4F is an anti-inflammatory, apolipoprotein A-I (apoA-I)-mimetic peptide that is active in vivo at nanomolar concentrations in the presence of a large molar excess of apoA-I. Physiologic concentrations ( approximately 35 microM) of human apoA-I did not inhibit the production of LDL-induced monocyte chemotactic activity by human aortic endothelial cell cultures, but adding nanomolar concentrations of 4F in the presence of approximately 35 microM apoA-I significantly reduced this inflammatory response. We analyzed lipid binding by surface plasmon resonance. The anti-inflammatory 4F peptide bound oxidized lipids with much higher affinity than did apoA-I. Initially, we examined the binding of PAPC (1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine) and observed that its oxidized products bound 4F with an affinity that was approximately 4-6 orders of magnitude higher than that of apoA-I. This high binding affinity was confirmed in studies with defined lipids and phospholipids. 3F-2 and 3F(14) are also amphipathic alpha-helical octadecapeptides, but 3F-2 inhibits atherosclerosis in mice and 3F(14) does not. Like 4F, 3F-2 also bound oxidized phospholipids with very high affinity, whereas 3F(14) resembled apoA-I. The extraordinary ability of 4F to bind pro-inflammatory oxidized lipids probably accounts for its remarkable anti-inflammatory properties.

Treatment with an apolipoprotein A-1 mimetic peptide in combination with pravastatin inhibits collagen-induced arthritis.

To evaluate the therapeutic potential of an apolipoprotein A-1 (apoA-1) mimetic peptide, D-4F, in combination with pravastatin in collagen-induced arthritis (CIA), syngeneic Louvain rats were immunized with type II collagen and randomized to vehicle control, D-4F monotherapy, pravastatin monotherapy, or D-4F + pravastatin combination therapy. Clinical arthritis activity was evaluated and radiographs, type II collagen antibody titers, cytokine/chemokine levels, and HDL function analysis were obtained. There was significant reduction in clinical severity scores in the high and medium dose D-4F + pravastatin groups compared to controls (p< or =0.0001). Reduction in erosive disease occurred in the medium/high dose combination groups compared to non-combination groups (p< or =0.01). Favorable changes in cytokines/chemokines were noted with treatment, and response to combination D-4F/pravastatin therapy was associated with improvement in HDL's anti-inflammatory properties. Combination D-4F/pravastatin significantly reduced clinical disease activity in CIA, and may have dual therapeutic potential in other autoimmune diseases with increased cardiovascular morbidity and mortality.

Apo A-1 mimetic peptides as atheroprotective agents in murine models.

The mouse has proven to be an excellent model for testing apolipoprotein mimetic peptides as agents to treat a variety of vascular inflammatory conditions including atherosclerosis, cognitive dysfunction associated with arteriole inflammation, chronic rejection of transplanted hearts, and scleroderma. The mechanism of action appears to relate to the ability of these peptides to preferentially bind pro-inflammatory oxidized lipids and is independent of the chirality of the peptides since peptides synthesized from either D- or L-amino acids appear to be equally effective.

Lipoprotein inflammatory properties and serum amyloid A levels but not cholesterol levels predict lesion area in cholesterol-fed rabbits.

Rabbits on a 1% cholesterol diet received injections of vehicle with or without D-4F or L-4F. After 1 month, the percent of aorta with atherosclerotic lesions was 24 +/- 15% (vehicle), 10 +/- 6% (D-4F) (P < 0.01 vs. vehicle), and 13 +/- 9% (L-4F) (P < 0.05 vs. vehicle). Inflammatory indexes for HDL and LDL were determined by measuring monocyte chemotactic activity after adding rabbit lipoproteins to human endothelial cells. HDL-inflammatory index (HII) and LDL-inflammatory index (LII), respectively, were 1.39 +/- 0.24; 1.35 +/- 0.29 (vehicle), 0.67 +/- 0.26; 0.63 +/- 0.38 (D-4F) (P < 0.001 vs. vehicle), and 0.67 +/- 0.2; 0.68 +/- 0.32 (L-4F) (P < 0.01 vs. vehicle). Serum amyloid A (SAA) levels were 95 +/- 39, 8 +/- 22, and 7 +/- 19 mug/ml, respectively, for vehicle, D-4F, and L-4F (P < 0.001 vs. vehicle). There was no correlation between lesion area and total plasma or HDL-cholesterol levels. In contrast, there was a positive correlation with HII, LII, and SAA (P = 0.002, P = 0.0026, P = 0.0079, respectively). HII correlated closely with SAA levels (r = 0.6616; r(2) = 0.4377, P < 0.0001). Thus, HII, LII, and SAA are better predictors of lesion area than are total plasma or HDL-cholesterol levels in cholesterol-fed rabbits.

High-density lipoprotein: antioxidant and anti-inflammatory properties.

The ability of high-density lipoprotein (HDL) to promote cholesterol efflux is an important component of its ability to protect against cardiovascular disease. In addition, the anti-inflammatory properties of HDL are important as well. As part of the innate immune system, HDL appears to have evolved to increase inflammation in the presence of an acute phase response but to inhibit inflammation in the absence of an acute phase response. In a study of humans with coronary heart disease, it was found that the patients who had proinflammatory HDL prior to statin therapy (and half of them despite a profound decrease in plasma lipids following statin therapy) continued to have proinflammatory HDL. Anti-inflammatory HDL was effective in promoting cholesterol efflux whereas proinflammatory HDL was relatively weak in its ability to promote cholesterol efflux. Oxidative alterations of the main protein of HDL, apolipoprotein A-I, impaired its capacity to promote cholesterol efflux from monocyte macrophages. Therefore, HDL composition, structure, and function appear to be more crucial than HDL cholesterol concentrations in determining risk for cardiovascular disorders.

Peptide Mimetics of Apolipoproteins Improve HDL Function.

Over the past decade evidence has accumulated that suggests that the anti-inflammatory properties of HDL may be at least as important as the levels of HDL-cholesterol. The recent failure of the torcetrapib clinical trails has highlighted the potential differences between HDL-cholesterol levels and HDL function. Agents to improve HDL function including HDL anti-inflammatory properties provide a new therapeutic strategy for ameliorating atherosclerosis and other chronic inflammatory conditions related to dyslipidemia. Seeking guidance from the structure of the apolipoproteins of the plasma lipoproteins has allowed the creation of a series of polypeptides that have interesting functionality with therapeutic implications. In animal models of atherosclerosis, peptide mimetics of apolipoproteins have been shown to improve the anti-inflammatory properties of HDL, significantly reduce lesions and improve vascular inflammation and function without necessarily altering HDL-cholesterol levels. Some of these are now entering the clinical arena as interventions in pharmacologic and pharmacodynamic studies.